Animal Models in Preclinical Evaluation of Sanofi Pasteur ... · 12 Guidelines WHO n°872, 1998,...
Transcript of Animal Models in Preclinical Evaluation of Sanofi Pasteur ... · 12 Guidelines WHO n°872, 1998,...
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Animal Models in Preclinical Evaluation of sanofi pasteur Flavivirus Vaccines
Dengue Tetravalent Vaccine Application Jean LANG M.D., Ph.D.
aVP R&D sanofi pasteur
Dengue R&D Program Leader
Fondation Merieux – Annecy- October 11th 2011
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Dengue Disease: A Major Public Health Concern
●Mosquito-borne disease
● 2nd most widespread tropical
disease after Malaria; 1st in
Latin America and Asia Pacific
●Almost 50% of world population
is at risk
● Each year:
● 230 million infected
● 2 million severe disease
(90% children)
● 25 000 deaths
● Increasing geographic spread
and burden of disease (1)Beatty M et al. AM J Trop Med Hygiene 81, 5:231 2009
(2) WHO 2009
Global Prevalence of
Dengue(1)(2)
Areas infested with Aedes aegypti
Areas with Aedes aegypti and
dengue epidemic activity
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Dengue Disease Prevention: High Unmet Medical Need
* World Health Organization, 2009 Fact Sheet #117
● Tetravalent dengue
vaccine
● Compliance with
WHO guidelines
● Will be made
available to
endemic areas first
Sanofi Pasteur
● Prevention through
vaccination is
an important
objective*
● Guidelines for
clinical evaluation,
production, quality
control, and
antibody testing
WHO
● No effective drug
treatments
● No effective
prevention
Treatment
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Developing a Safe and Effective Tetravalent Vaccine against Dengue
FOR 4 SEROTYPES SIMULTANEOUSLY
A Balancing Act
ATTENUATED POTENT
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Innovative Approach Designed to Maximize Safety
YF 17D Vaccine Dengue Virus
● YF vaccine: Live attenuated
●High genetic stability
●Dengue virus: Wild Type
● Serotype differences linked to
protein differences at surface
● Envelope E proteins trigger
production of neutralizing antibodies
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CYD Dengue Vaccine: Tetravalent Combination of Chimeric LAV
● Four genetic constructs are created, one for each serotype
●All are based on same YF 17D backbone
● Insertion of E and prM genes, isolated from each serotype
prM E
prM E
prM E
prM E
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2
3
4
C YFV17D Non-Structural genes
CYD4
CYD1
CYD2
CYD3
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T
B
Clinical
Immunogenicity /
Safety
Mosquito
vectors
DCDC
In vitro models
• PBMCs, DCs,
• Monocytes,
• Endothelial cells
• Hepatic cells/ lines
Mo
• Infectivity
• Tropism
• Phenotype modification
• Cytokines induction
• Viscerotropism
• Abs
• CMI
• Viremia
• Safety
• Protection
• Replication
• Transmission
• Genetic stability
TB
Animal models
• Monkeys
• Mice
• Hamsters
• Immunogenicity
• Viremia
• Safety
• Protection
• Neurovirulence
• Viscerotropism
• Biodistribution
• Interferences
Guy et al Vaccine. 2010, 2011
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Vaccine Development Pathway
=> GMO risk assessment
=> Toxicology assessment
=> Vaccine characterization
=> Immunological characterization
=> New Ag/Techno identification
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PreClinical Dengue Vaccine Development
Mouse Monkey
Neurovirulence
Immunogenicity
Viremia
Neurovirulence
Immunogenicity
Man
Reactogenicity
Immunogenicity
Protection
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● Macaques: Rhesus and cynomolgus
● Due to species availability, cynomologus were the most used
● Same design for most of protocols. Dosing scheme and end-points tailored based on study objectives
● Same dose as clinical trials (passage, formulation): 3 to 5 log
● 1 to 3 SC administrations, usually 8 wks apart
● 4 to 6 flavivirus-negative monkeys per group
• Animal screening prior to the test. Depend on the origin
● Ethical review process includes species justification
Primate species and Immunology protocols
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General Toxicology
● Repeated dose-toxicity study, GLP
● Study Objective: to assess the systemic safety and the local tolerance of CYD1-4 vaccine candidates
● Monovalent vs tetravalent
● Well tolerated
● No synergistic effect when Tetravalent
● No major direct or indirect toxic effects
● No concerns identified
● Supportive of the clinical trials
0 3 10 28 31 38 49 56 66 77 - days -
Blood analysis Necropsy & histology
CYD TV
+
Control
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● Guidelines
● WHO n°872, 1998, A4.1.3, Monkey safety test (neurotropism test) or Eur.
Ph. 5th Ed: Yellow fever vaccine (live)
● Comparator is parental YF-17D
● 0.25 ml in frontal lobe (~5 Log CCID50)
● Every CYD serotype at master seed level
● Mono and tetravalent CYDs are less neurovirulent than YF17D
● Target score ranged from 0.03 to 0.15 vs 1.1 for YF-17D
● Suckling mouse model (4 days old) is more discriminating
● Done at Master & Working seed levels
● Less Neuroviruent than licensed YF 17D 204 licensed vaccine
Neurotropism
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ChimeriVaxTM-DENTV Monkey safety test Histopathology : Group mean histological lesion scores in Brain & spinal cords -
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N
0.0018 0.0015 0.0023 P value (ANOVA)
0.39 0.35 0.43 YF - VAX®
0.04 0.03 0.05 ChimeriVax ™ - DENTV
Combined Discriminator areas
Target area
Mean ( + SD) Test article
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N
0.0018 0.0015 0.0023 P value (ANOVA)
0.39 0.35 0.43 YF - VAX®
0.04 0.03 0.05 ChimeriVax ™ - DENTV
Combined Discriminator areas
Target area
Mean ( + SD) Test article
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● Study objectives: assess the distribution, the persistence or elimination,
as well as the shedding of the vaccine
● Scheduled protocol
● One SC administration with final formulation
● 3 time points: peak of viremia, end of viremia, late point
● About 20 tissues per animal will be tested (RT-PCR): liver, lung, heart, blood,
kidney, testes
● Viral shedding: faeces, urine, saliva, injection site, brain, lymphoid tissues
● Results
● Limited distribution/replication mainly at the injection site and draining lymph
nodes and in a limited number of other tissues such as liver or lymphoid
tissues, without viral persistence.
● No associated changes in clinical pathology parameters, and no findings at the
histopathology examination
Biodistribution, viral shedding
Toxicity, biodistribution and shedding of sanofi pasteur live attenuated, tetravalent dengue vaccine in
cynomolgus monkeys. Ravel G, Mantel N, Lang J, Gould S. Eurotox
(Meeting of European Societies of Toxicology), Paris, France, 2011
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● DEN4 strain 1228 and Asibi used as controls
● Model characterization
● Background data
● Comparison of pathology and biodistribution with CYD and vaccinal
YF17D
● Little (YF17D) to no infection foci (CYD), no pathology
● Lack of pathology associated with DEN4 strain 1128
● No clinical signs, or biomarker of infection, enlarged spleen,
adenomegalia
Liver Tropism Monkey Model
Lefeuvre A. Thèse Univ Lyon, 2005
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In vitro hepatotropism summary
● Viscerotropism is characteristic of wild-type flavivirus
infection in humans
● Human cell lines have been used to evaluate in vitro
hepatotropism of CYD candidates
● CYD do not replicate or at very low titers in HepG2 cells,
compared YF17D
● Similar study with same conclusions performed at Acambis on
HepG2 and ThLe cells
● On going: evaluation of primary hepatocytes derived from
human stem cells
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● Negative predictivity of primate model
● ‘Hot’ strains in NHP are hot in man
● Low viremia strain does not predict attenuation in man
● Main criteria for in vivo attenuation evaluation is viremia
(one marker of viscerotropism)
● Similar immune response (% of response after
immunization, mean titer) but with different serotype
dominance
● Lack of disease with wild-type DEN viruses
Human Predictivity of Monkey Models
Sanchez V et al. Vaccine, 2006
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● Viremia
● Short lasting
• Onset: Day 6, mean duration: 2-3 days
● Low level
• mean peak level: 1 to 2 log pfu/ml
● Almost only with CYD4. Interaction between serotypes
● Lower or no viremia after 2nd immunization
● Neutralizing Ab (PRNT)
● Variable according to serotype (4 > 1 > 2-3)
● Boost is required
● Convenient model to test alternate schedule of immunization
● One year boost induces strong responses against all 4 serotypes
● Challenge models
● Protective immunity against all 4 serotypes 6 months post immunization
● Viremia as marker of protection; no detectable viremia
Monkey Immunology assessment
Guirakhoo F et al. J Virol, 2004, Guy et al 2009
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● Interaction between serotypes
● Different viremia if monovalent or tetravalent
● Most predominant serotype might change depending on the
species
• In NHP, CYD4 > 1 > 2-3
• In Man, CYD4 > 2-3 > 1
● But no synergistic effects
● Tetravalent vaccine has the same pattern of the ‘hottest’
monovalent (infectivity pattern)
Monkey Model & Interference
Guy B et at. AJTMH, 2009
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● In most cases, KO mouse models (type I/II IFNR, such as A129
or AG129)
● Non immunocompetent mice, unsuitable to assess
immunogenicity/protection
● Humanized mice
● Complex, human donor variability
● WHO TRS932 Recommendation (part B.4.3)
● “At present, the AG129 mouse seems most suitable for safety
studies, but National regulatory authorities should be aware of
the pitfalls of interpreting results, since these animals do not
possess an intact innate immune response. For this same
reason, as mentioned earlier, it would not be advisable to use
AG129 mice for classic toxicology studies”
Mouse models for preclinical dengue vaccine evaluation
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Conclusions : Key Elements of the sp Dengue Vaccine Non-Clinical Development Risk Management Strategy
The vaccine is not transmitted
by mosquitoes
Reversion to virulence
is almost impossible
?
Natural recombination is highly unlikely
?
X
Gene transfer is impossible
Excellent genotypic and phenotypic stability
Tetravalent vaccination will address the ADE risk
The vaccine neutralizes all
circulating strains tested so far
Vaccine. 2010 Jan 8;28(3):632-49
From research to phase III: Preclinical, industrial and clinical development of the Sanofi Pasteur tetravalent dengue vaccine.
Guy B, Barrere B, Malinowski C, Saville M, Teyssou R, Lang J. Vaccine. 2011 Sep 23;29(42):7229-41
Mantel N, Girerd Y, Geny C, Bernard I, Pontvianne J, Lang J, et al., Genetic stability of a dengue vaccine based on chimeric yellow fever/dengue viruses. Vaccine
2011
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Presentation Contributors
● Sanofi pasteur
● Bruno Guy, Director, Discovery
● Guillaume Ravel, Director, Non Clinical Safety
● Veronique Barban, Director, Virology
● Farshad Guirakhoo, Director, external RD
● Thierry Decelle, Director, Animal Science & Welfare